Human cytomegalovirus (HCMV) is a species-specific herpesvirus that causes morbidity and mortality in immunocompetent and, especially, in immunocompromised hosts. HCMV disease can involve multiple organ systems and a critical feature of infections is, therefore, the hematogenous dissemination of the virus. We have documented that monocytes/macrophages are likely the principal cell types responsible for HCMV spread to peripheral organs following primary infection and are key reservoirs for viral persistence, suggesting these cells are the source of the initial spread to organ tissue following primary infection and for the long-term viral persistence observed in the host. The viral strategy of utilizing monocytes and macrophages for viral dissemination and persistence links HCMV infection to viral-mediated pathogenesis. Our work and the work of others suggest the following model for viral spread. Monocytes are infected in the blood, although not productively at the time of initial infection, and are induced by viral binding to cognate receptors to extravasate int various tissues. There they differentiate into long-lived macrophages, which support replication of the original input virus, allowing for viral persistence in target organs. The ability to drive monocyte extravasation and monocyte-to-macrophage differentiation appears to be an essential function of the virus. Our recent studies documented for the first time that the epidermal growth factor receptor (EGFR) and ?1 and ?3 integrins are bona fide viral receptors on monocytes and that their engagement during viral binding (via glycoprotein B (gB) and the gH/gL/UL128-131 complex, respectively) is the key trigger that directly links infection and entry of monocytes to the distinct functional changes associated with viral persistence. These identified viral receptor/ligand interactions promote unique biochemical and molecular changes in monocytes focused on altered/enhanced motility, survival and differentiation that are not mimicked by other viruses/pathogens or known monocyte-activating agents. These molecular studies are consistent with our new bioinformatics-based meta-analyses showing that HCMV-infected monocytes exhibit a distinct gene expression pattern when compared to that observed with monocytes treated with a variety of cytokines/chemokines and pathogens and that this unique macrophage profile remains for the course of infection (from weeks to months). For the virus, the culmination of this HCMV-directed biological reprogramming of monocytes is productive infection. Our new data identifies that viral entry occurs via a modified endocytosis-like process, and that viral trafficking (the mature particle is retained for days in various vesicles in the cytoplasm prior to eventual nuclear translocation ~4 days post infection), uncoating and gene expression profiles in infected monocytes/macrophages is distinct from that observed during infection of other cell types. Furthermore, our new data since the original submission now shows strongly that the delayed trafficking, uncoating and nuclear translocation is directed by the specific engagement of EGFR and integrins during viral binding. From our data, viral receptor/ligand interactions and the ensuing signaling emerges as the molecular convergence point where the needs of the virus (i.e. entry, nuclear translocation) meet the specific functional changes in monocytes (i.e. motility, survival, and differentiation) required for the use of these cells as vessels for spread and persistence. Based on these new findings, we hypothesize that a unique combination of HCMV-mediated receptor/ligand interactions triggers the distinct changes in infected monocytes that promote viral persistence. To test our hypothesis, we propose the following specific aims - 1) to examine how receptor/ligand-mediated signaling triggers the early biological changes associated with HCMV infection; 2) to investigate the regulation by which HCMV-induced signaling promotes viral trafficking in infected monocytes/macrophages; and, 3) to determine the mechanisms for how receptor/ligand-mediated signaling induces differentiation of infected monocytes. The results from this proposed study will provide insight into how HCMV infection initiates the molecular changes in infected monocytes and macrophages required for viral spread and persistence. Because we are also providing a new understanding about the biochemical and molecular processes that may control myeloid cell biology, our studies have the potential to have broad immunological implications for the understanding of myeloid cells. Furthermore, the decoding of the key regulatory pathways required for changes and infection of monocytes will provide molecular insight into the underlying causes of HCMV pathogenesis, as well as identify new potential target pathways for therapeutic intervention.